Stromal and thylakoid-bound ascorbate peroxidases are produced by alternative splicing in pumpkin

A cDNA encoding stromal ascorbate peroxidase (sAPX) was isolated using poly(A)+ RNA of pumpkin cotyledons by RT-PCR. The cDNA encodes a polypeptide with 372 amino acids and shares complete sequence identity with pumpkin thylakoid-bound ascorbate peroxidase (tAPX), except for the deletion of a putative membrane spanning region located in the carboxy domain of tAPX. Southern blot hybridization and analysis of intron structure indicated that mRNAs for sAPX and tAPX, whose suborganellar localizations in chloroplasts are different, are produced by alternative splicing. Immunoblot analysis showed that the accumulation of sAPX and tAPX was differently regulated during germination and subsequent greening of pumpkin cotyledons.